Non-luminescent substrate

ABSTRACT

A substantially non-reflective, low or non-fluorescing substrate comprising a phase-inversion support and a plurality of opaque solids that are substantially chemically non-reactive with the phase inversion support and of a size sufficient to be partially or completely contained within, or intimately bound to, the phase inversion support. Methods of making and using the substantially non-reflective, low or non-fluorescing substrate are also disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of commonly owned U.S.Provisional Patent Application Ser. No. 60/216,229 of Andreoli, filedJul. 5, 2000, entitled “IMPROVED NON-LUMINESCENT SUBSTRATE,” thedisclosure of which is herein incorporated by reference to the extentnot inconsistent with the present disclosure.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates to an improved fluorescent-quenchingsubstrate comprising a phase inversion support associated with aplurality of opaque solids that are substantially non-reactivechemically with the phase inversion support and that are of a sizesufficient to be partially or completely contained within, orirreversibly bound to, the phase inversion support. Such substrate mayadvantageously be employed in bioaffinity assays, including immunoassaysand nucleic acid binding assays, which utilize luminescent tags, such asfluorescent tags. Such substrate further has use as filtration media toefficiently remove organic and/or inorganic material from fluids.

A great variety of assay systems have been developed to detect thepresence and concentration of analytes in samples. For example,bioaffinity and enzymatically-activated catalysis reactions are widelyused in medicine and science to analyze biological samples to detect andquantitize biological materials of concern. Many of these assay systemsdepend upon the binding of one chemical entity with the material ofconcern (or a modified form thereof) and detection of the conjugate,e.g., antigen-antibody, nucleic acid strand to complementary nucleicacid strand (“hybridization”), and protein-ligand conjugates. Theconjugate is typically detected by way of a label providing a detectablesignal which is attached to one or more of the binding materials. Theconjugate is frequently quantitated by first determining the amount oflabel in the free and bound fractions, and then calculating the amountpresent using an algorithm and a set of standards to which the samplesare compared.

The most common labels used in analyte binding assays are radioisotopesand luminescent compounds. Radioisotopes (Isotopic labeling) proffersconsiderably better detection in certain analyte systems thanluminescent labeling. For example, the most sensitive methods fordetecting nucleic acids typically involve the use of isotopic labeling,often involving radiolabelling with ³²P Luminescence is induced byenergy transfer and refers to light emission that cannot be attributedmerely to the temperature of the emitting body. Luminescent labels canbe made to luminesce through photochemical (so-called,“photoluminescence”), chemical (so-called, “chemiluminescence”) andelectrochemical (so-called, “electrochemiluminescence”) means.Photoluminescence, which includes fluorescence and phosphoresence, is aprocess whereby a material is induced to luminesce when it absorbselectromagnetic radiation such as visible, infrared or ultravioletradiation. Chemiluminescence refers to luminescence occurring as aresult of a chemical reaction without an apparent change in temperature.Electrochemiluminescence refers to luminescence occurring as a result ofelectrochemical processes.

In localizing particular sequences within genomic deoxyribonucleic acid(“DNA”), a transfer technique described by Southern is typicallyemployed. DNA is digested, often using one or more restriction enzymes,and the resulting fragments are separated according to size byelectrophoresis through a gel. Conventionally the DNA is then denaturedin situ and transferred from the gel to a solid support, the relativepositions of the DNA fragments being preserved during and after thetransfer to the solid support. The DNA attached to the solid support isthen hybridized to radiolabelled DNA or ribonucleic acid (“RNA”), andautoradiography is used to locate the positions of bands complementaryto the probe.

For many years, immobilization and hybridization of denatured DNA wascarried out almost exclusively using nitrocellulose as a solid support.As time progressed, however, it became apparent that nitrocellulose wasa less than an ideal solid-phase hybridization matrix, as nucleic acidsare attached to the nitrocellulose support by hydrophobic, rather thanby covalent interactions, and the nucleic acids are released slowly fromthe matrix during hybridization and washing at high temperatures. Toovercome this problem, charge-modified cellulose supports, including DBM(diazobenzyloxymethyl)-cellulose and APT-cellulose, were introduced inthe early 1980's to provide improved nucleic acid binding. Thesematrices, however, like nitrocellulose itself, also suffer from asignificant disadvantage in that they become brittle when dry and cannotsurvive more than one or two cycles of hybridization and washing, i.e.,“reprobing.”

Extensive use today is made of polyamide matrices, in particular nylonmatrices, as solid support for immobilization and hybridization ofnucleic acids. Various types of nylon are known to bind nucleic acidsirreversibly and are far more durable than nitrocellulose. As nucleicacids can be immobilized on nylon in buffers of low ionic strength,transfer of nucleic acids from gels to a nylon matrix can be carried outelectrophoretically, which may be performed if transfer of DNA bycapillary action or vacuum is inefficient. Two basic types of nylonmembranes are commercially available, unmodified nylon andcharge-modified nylon. Charge-modified nylon is preferred for transferand hybridization as its increased positively-charged surface has agreater capacity for binding nucleic acids (See, e.g., U.S. Pat. No.4,473,474, the disclosure of which is herein incorporated by reference).Nylon membranes must be treated to immobilize the DNA after it has beentransferred, as by way of thorough-drying, or exposure to low amounts ofultraviolet irradiation (254 nm).

While polyamide matrices have found considerable use in isotopic assaysystems, such matrices have not found widespread use in fluorescentassay systems. This is likely due to the fact that fluorescent assaysystems employing polyamide substrates demonstrate less than desirablesensitivity. Such reduction in sensitivity has been attributed primarilyto two factors—background fluorescence produced by the nylon itself, andlight scattering by solid materials in contact with the reaction media(such as substrates to which reactants are attached, or walls of thecontainers in which measurements are made). Polyamides, such as nylon,show light-stimulated endogenous fluorescent emissions and lightreflection which can coincide with the range of UV-visible wavelengthsemitted from fluorophore-tagged analytes. When light in the excitationwaveband causes fluorescence of the support material, interference withdetection occurs if the emission waveband of the fluorophore overlapsthe same.

While isotopic assays on the whole are very sensitive, they suffer froma number of disadvantages. Primarily, use of any radioisotopeautomatically invokes health concerns and a host of regulatory dutieswith respect to waste disposal, safety, handling, reporting andlicensing. While present luminescent assays proffer an alternative toisotopic labeling, the sensitivity of such assays is still not within arange desired by many in the biomedical, genetic research and drugdiscovery communities. Additionally, isotopic labeling cannot be used inmultiplex assays, in which two or more nucleic acid probes which havebeen separately labeled each with their own unique colored luminescentlabel can be simultaneously hybridized, then simultaneously detected onan array of bound nucleic acid targets affixed to the polymericsubstrate. Multiplexing saves significant cost and time when compared tothe traditional steps of stripping and reprobing when performingmultiple queries on a given array of targets. Multiplexing also reduceserror and signal degradation that is associated with multiplereprobings.

U.S. Pat. Nos. 4,837,162 and 4,921,878 to Rothman et al., disclose adye-modified polyamide material for use in luminescent assays which issaid to both reduce the background fluorescence due to the polyamide, aswell as light scattering by solid materials in contact with the reactionmedia. By reducing such properties, it asserted that such polyamidesubstrates allow improved detection of fluorescent emissions fromfluorophore-tagged analytes as compared to untreated polyamidesubstrates. These patents disclose dyeing the polyamide material with areactive dye, that is, a dye that contains a functional group thatchemically reacts with the material being dyed (See, Column 6, Lines54-57, of U.S. Pat. No. 4,921,878), having an absorbance spectrumselected to overlap the excitation and/or emission waveband of lightgenerated by the polyamide substrate. It is asserted that acid-reactivedyes capable of integrally binding to the polyamide do not adverselyaffect the properties of the polyamide substrate. Acid-reactive dyes ofthe azo class are said to be particularly useful and to be readilymanipulated to absorb light in the necessary waveband. Non-metallic acidreactive dyes are said to be preferred.

While the dyed-polyamides disclosed in U.S. Pat. Nos. 4,837,162 and4,921,878 have been known for over a decade, such polyamides have notfound widespread acceptance in luminescent assay systems. The failure ofsuch substrates to dominate the market may relate to the less thandesirable fluoresecence quenching that has been able to be producedfollowing the disclosures of U.S. Pat. No. 4,837,162. It may also relateto the difficulty in identifying dyes that are both chemically-reactivewith respect to the nylon and chemically non-reactive with typicalanalytes of interest. It is also possible that the dyes specified inthese references interfere with binding of the biomolecules with thenative nylon surface, either by creating an unfavorable surface foradsorption of the biomolecule, or directly competing with nylon foradsorption. One possible outcome of this competition is the creation ofa biomolecule:azo dye complex which is less stable and more easilyextracted and lost from the nylon surface, thus detrimentally affectingthe analysis.

The biomedical and scientific communities would eagerly use fluorescentassays, as opposed to isotopic assays, if the detection sensitivity offluorescent assays can be enhanced without increasing the potential forundesired chemical reactions. While sensitivity can be increased if thesubstrate on which fluorescent assays are performed does not fluoresceupon such exposure, isolation of such substrates having widespreadusefulness (with respect to numerous analytes) has so far eluded theart. There is a need, therefore, for improved substrates for use inluminescent assays which lead to greater sensitivity for detectinganalytes in a sample.

SUMMARY OF THE DISCLOSURE

The present application discloses a substantially non-reflective, low ornon-fluorescing (from about 250 nm to about 770 nm), substratecomprising a phase-inversion support and a plurality of opaque solidsthat are chemically non-reactive with the phase inversion support and ofa size sufficient to be partially or completely contained within, orirreversibly bound, to the phase inversion support. Such substratesignificantly improves detection in luminescent assay systems withrespect to the detection of a wide-variety of analytes, and enables theuse of a wide variety of fluors as labeling moieties. Further disclosedis an improved fluid filtration polyamide substrate. Such polyamidesubstrates comprise a polyamide support impregnated (fully orpartially), coated, or surface-bound, with light absorbing opaquesolids, such as pigments, fillers and extenders. Such polyamidesubstrates can be used as solid support matrices in bioassays or in thefabrication of objects such as reaction containers, as well as filtersfor removing organic and/or inorganic materials.

In one representative embodiment, there is disclosed a polyamidesubstrate comprising a polyamide support with opaque solids, such as apigment, as for example, carbon particles, interspersed therein. Thepolyamide support may comprise nylon-66. Preferably the carbon particlesare amorphous, and advantageously comprise carbon black. The carbonparticles preferably compose at least about 0.002% by weight of thepolyamide substrate. The carbon particles may be impregnated wholly orpartially within the polyamide support or may be distributed over one ormore surfaces of the polyamide support. Preferably distribution of thecarbon particles is substantially uniform. And more preferably, thecarbon particles are uniformly impregnated throughout the polyamidesupport such that they are entirely encapsulated within the supportleaving the polyamide surface chemically functional, and relativelyunaffected by the carbon particles. The latter has been found to providefor superior binding of nucleic acids, and their expressed products,proteins and other biomolecules.

In another representative embodiment, there is provided an opticallypassive polyamide substrate comprising a polyamide support and opaquesolids that are chemically non-reactive with the phase inversion supportand of a size sufficient to be partially or completely contained within,or irreversibly bound, to the phase inversion support, the opaque solidsbeing in a weight ratio such that said substrate is capable of absorbinglight at substantially all wave lengths from about 250 to about 770 nm,more preferably from about 300 to about 700 nm, that impinge upon it.The polyamide substrate may be microporous, and may be in the form of amembrane. In one represenatative embodiment, it is presently preferredthat the polyamide substrate have a reflectance of no more than 50% ofincident light at any wavelength within said range of wavelengths. It isalso presently preferred that the polyamide substrate is hydrophilic andskinless.

In yet another representative embodiment, there is provided an assay inwhich the presence or quantity of an analyte is being detected byfluorescence at an emission waveband of light that results from theexcitation of a fluorescent signal label on a polyamide support by anexcitation waveband of light, the improvement comprising: providing assaid polyamide support an optically passive polyamide prepared byinterspersing carbon particles into a polyamide support in such a manneras to substantially quench fluorescence due to the polyamide support atthe excitation waveband or emission waveband, or both.

In still another representative embodiment, there is provided asubstrate comprising: a phase-inversion support; and a plurality ofopaque solids that are substantially chemically non-reactive with thephase inversion support and intimately bound thereto, and/orpartially/completely contained within the phase-inversion support, thesubstrate having substantially reduced reflectance. Methods forpreparing such polyamide substrates are also disclosed.

In another representative embodiment, there is provided a substratewhich provides little fluorescence from about 300 nm to about 700 nmcomprising: a phase-inversion support; and a plurality of opaque solidsthat are substantially chemically non-reactive with the phase inversionsupport and intimately bound to, and/or partially/completely containedwithin, said phase-inversion support, the substrate having substantiallyreduced reflectance.

In one representative method, a substrate is prepared comprising apolyamide support impregnated with, and substantially containingtherein, a plurality of opaque solids, such as carbon particles, suchthat substantially all of the polyamide surfaces are chemically andfunctionally available for binding of analyte; said method comprisingthe steps of: formulating a casting dope comprising a solvent,non-solvent, opaque solids and polyamide(s); mixing and blending thecasting dope to cause dissolution of the polyamide and opaque solids;causing an opaque solid-filled phase inversion of the casting dope;casting and then quenching a portion of the opaque solid-filled castingdope to fabricate a phase inversion substrate; and drying the substrate.

In another representative method, a substrate is prepared comprising theacts of: formulating a dope comprising a solvent, at least onenon-solvent, opaque solids and at least one phase-inversion polyamide;mixing and blending the dope to cause dissolution of the polyamide andopaque solids in the dope; and producing an opaque solids-filled phaseinversion membrane from the dope.

BRIEF DESCRIPTION OF THE DRAWINGS

The above description, as well as further objects, features andadvantages of the present disclosure will be more fully understood withreference to the following detailed description when taken inconjunction with the accompanying drawings, wherein:

FIG. 1 is a fluorescence spectral synchronous kinetic scan, using aconstant Stokes shift set at 25 nm (i.e., detector frequency centered ona wavelength which is 25 nm higher than the excitation light frequencyover the wavelengths scanned), of nylon-66 (top curve), and nylon-66impregnated with carbon black at a weight ratio (nylon-66:carbon black)of 10:1, 12:1, and 15:1 (bottom curves).

FIG. 2 is a fluorescence spectral synchronous kinetic scan, using aStokes shift set at 25 nm, of nylon-66 (top curve), and nylon-66impregnated with carbon black at a weight ratio (nylon-66/carbon black)of 21:1 and 31:1 (bottom curves).

FIG. 3 is a southern blot of bacteriophage lambda DNA digested with HindIII transferred to nylon membranes having varying amounts of carbonblack.

FIG. 4 is a cross-sectional drawing of a preferred opaque solid, carbonparticles, dispersed within the matrix of a polyamide phase inversionmicroporous membrane support.

FIG. 5 is a scanning electron micrograph of a polyamide phase inversionmicroporous membrane support, with and without, incorporation into itsmatrix of a preferred opaque solid, carbon particles.

DETAILED DESCRIPTION

The present disclosure overcomes many of the problems associated withthe less than desirable solid substrates used in analyte assaysemploying fluorescent labeling, and provides a product useful in anumber of other applications, including filtration.

There is disclosed a phase-inversion substrate impregnated (fully orpartially), coated, or surface-bound (or combination of the same), withopaque solids that are non-reactive with the phase inversion support andof a size sufficient to be partially or completely within, orirreversibly bound, to the phase inversion support. In a presentlypreferred representative embodiment, the substrate is a membrane, whichmay or may not carry charge. When employed in analyte assays which arebased on luminescent labeling, substrates containing such opaque solidshave been found to allow significantly enhanced detection of numerousanalytes under many conditions. Such substrates have been seen toproduce significantly less intrinsic fluorescence and light-scatteringthan polyamide substrates lacking the opaque solids.

By “phase inversion support” it is meant a polymeric support that isformed by the gelation or precipitation of a polymer membrane structurefrom a “phase inversion dope.” A “phase inversion dope” consists of acontinuous phase of dissolved polymer in a good solvent, co-existingwith a discrete phase of one or more non-solvent(s) dispersed within thecontinuous phase. The formation of the polymer membrane structuregenerally includes the steps of casting and quenching a thin layer ofthe dope under controlled conditions to affect precipitation of thepolymer and transition of discrete (non-solvent phase) into a continuousinterconnected pore structure. This transition from discrete phase ofnon-solvent (sometimes referred to as a “pore former”) into a continuumof interconnected pores is generally known as “phase inversion.” Suchmembranes are well known in the art.

Typically, a phase inversion support is formed by dissolving thepolymer(s) of choice in a mixture of miscible solvent(s) andnon-solvent(s), casting a support pre-form, and then placing the surfaceof the support pre-form in contact with a non-solvent (liquid oratmosphere) diluent miscible with the solvent(s) (thereby precipitatingor gelling the porous structure).

By “opaque” it is meant displaying the property of not being pervious tovisible light. By “solid” it is meant a composition of matter that isnot entirely either a liquid or gas, or both. A “solid” may, or may not,have internal cavities or channels. A “solid” with an internal cavity orchannel may comprise a liquid or gas within the internal cavity orchannel. Solids may be formed in any variety of shapes, and have aplurality of textures as well. Roughly spherical solid particles can beadvantageously used. Such a solid particle may also have internalcavities or channels (i.e. a reticulated particle); the channelsproviding greater surface area or light absorptive characteristics. Inaddition, non-spherical solids such as fibers may also be advantageouslyused. A fiber is generally defined as a solid particle having an aspectratio of greater than 2 units of length per 1 unit of diameter. Fibersof considerable length may also be employed in the present disclosure,thus contributing properties unique to the combination of fiber andmembrane composite (i.e. improved strength) to the resultant structures.

By “intimately bound” it is meant that one substance is bound to anothersubstance in a manner that it is not easily dissociated from the othersubstance. As used in this application, “intimately bound” does notinclude binding, which is predominantly by means of a chemical bondbetween the one substance and the other substance.

A presently preferred phase inversion support comprises polyamides,organic polymers formed by the formation of amide bonds between monomersof one or more types. Particularly useful polyamides in the presentdisclosure are nylons. Nylons comprise aliphatic carbon chains, usuallyalkylene groups, between amide groups. The amide groups in nylons arevery polar and can hydrogen bond with each other, and are essentiallyplanar due to the partial double-bond character of the C—N bond. Nylonsare polymers of intermediate crystallinity, crystallinity being due tothe ability of the NH group to form strong hydrogen bonds with the C═Ogroup. Nylon typically consists of crystallites of different size andperfection. It is the amorphous content of nylons that adds a diffusescattering halo. Nylon 66, typically synthesized by reacting adipic acidwith hexamethylene diamine, is a particularly preferred nylon for thepresent disclosure. Nylon 66 will typically contain both fluorescent andphosphorescent species which can not be extracted by conventionalextraction techniques. These species are believed to be associated withthe presence of α-ketoimide structures formed by thermal oxidation ofthe molecular backbone of the polymer, and associated with, ororiginating from, aldo condensation products of cyclic enone dimer anddienone trimer of cyclopentanone; all of which are present in thepolymer as manufactured (See, Allen et al., Analysis of the Fluorescentand Phosphorescent Species in Nylon-66, Eur. Polym. J., 21(6), pp.517-526, 1985).

A carbon-polyamide substrate of the present application can be producedby coating the surface of, or impregnating, a polyamide support, such asa mesh, with carbon black.

The polyamide, such as nylon-66, could be produced with carbon blackmixed into a dope, such dopes as described in U.S. Pat. No. 3,876,738and/or U.S. Pat. No. 4,645,602, so as to form a carbon-black filledpolyamide microporous membrane substrate.

Polyamide substrates can be formed into planar solid supports,containers, and filters. Presently preferred polyamide substrates arereadily wettable by the liquids with which they are to be contacted, andare preferably hydrophilic. Presently preferred polyamide substrates arealso porous. The polyamide substrate may comprise a microporousmembrane. The substrate is preferably also skinless, that is, thepolymer organization does not change from the exterior surface to theinterior surface of the polyamide. Nylon-66 is a presently preferredpolyamide, particularly in the form of a skinless, hydrophilicmicroporous membrane.

It has been discovered by the present inventors that the fluorescence ofpolyamide materials, such as nylon, can be significantly reduced byaddition of opaque solids that are chemically non-reactive with thephase inversion support and of a size sufficient to be partially orcompletely contained within, or irreversibly bound, to the phaseinversion support, e.g., carbon particles. Amorphous carbon, such ascarbon black, a pigment, has been found to be a particularly usefulopaque solid. By the term “carbon black” it is meant an amorphous formof carbon produced commercially by thermal or oxidative decomposition ofhydrocarbons, and includes: (1) animal charcoal obtained by charringbones, meat etc.; (2) gas black obtained by incomplete combustion ofnatural gas; (3) lamp black, obtained by burning various fats, oils,resins, etc., under suitable conditions; (4) activated charcoal preparedfrom wood and vegetables.

Presented with numerous possibilities for quenching the fluorescence ofpolyamide materials, the present inventors have found that substantiallychemically non-reactive (with respect to the polyamide) opaque solids,such as carbon particles, may be physically associated with a polyamidesupport to allow efficient detection of numerous analytes inphotoluminescent binding assays (without interference from backgroundfluorescence). In particular, DNA binding during Southern Blot transferhas not been seen to be adversely affected by the presence of suchopaque solids such as carbon black. Enhanced detection of DNA and othernucleic acids may be evidenced when polyamide substrates are impregnated(partially or wholly), coated, or surface-bound, with such opaque solidswhen photoluminescence assays are used.

While any opaque solid that is non-reactive with the phase inversionsupport and of a size sufficient to be partially or completely containedwithin, or irreversibly bound to the phase inversion support which hasthe desired fluorescence quenching properties may be used, black solidsin particular, such as carbon-black, have been advantageously employed.Carbon-black absorbs energy thereby quenching the fluorescing backgroundproduced by a nylon-66 membrane. The simple chemistry of carbon black,once incorporated into the membrane, has not been found to interferewith nucleic acid binding assays, in particular with DNA binding duringSouthern Blot transfer.

Alternatively, it is believed that a suitable coating of pigment (bywhich it is meant a solid that reflects light or certain wavelengthswhile absorbing light of other wavelengths, without producingappreciable luminescence) either impregnated partially or mostly withinthe polymer matrix or properly and intimately bound to the surfaces(internal and external surfaces of the porous matrix) of such amicroporous membrane may also be employed; especially when it isdesirable to have the chemical functionality of the pigment availablefor interaction with analytes.

The present inventors have also discovered that activated carbon-coatedpolyamide substrates, in particular nylon substrates, and polyamidesubstrates having activated carbon partially encompassed therein (i.e.,having a portion of the activated carbon particles exposed on thesurface of the polyamide substrate) have been found to provide enhancedremoval of organic contaminants in drinking water as well as particleremoval. The increase in removal of organic contaminants from fluidswhich is evidenced using activated carbon-polyamide substrates, asopposed to nylon alone, or activated carbon alone, may be due to thegreater surface proffered when the activated carbon particles aredispersed among the polyamide support.

Now turning to FIG. 1, there is shown a fluorescence spectralsynchronous kinetic scan, using a Stokes shift set at 25 nm, of nylon-66(top curve), and nylon-66 impregnated with carbon black at a weightratio (carbon black:nylon-66) of 1:10, 1:12, and 1:15 (bottom curves).As evidenced, carbon black was seen to significantly reduce thefluorescence of nylon-66. The spectral kinetic scans of the 1:10, 1:12and 1:15 substrates were found to be within the background noise for theparticular equipment employed.

Now turning to FIG. 2, there is shown a fluorescence spectralsynchronous kinetic scan, using a detection stokes shift set at 25 nm,of nylon-66 (top curve), and nylon-66 impregnated with carbon black at aweight ratio (carbon black:nylon-66) of 1:21 and 1:31 (bottom curves).Even though carbon comprised a significantly lower percentage of theweight of the substrate as compared to the substrates of FIG. 1, carbonblack was seen to reduce significantly the fluorescence of nylon-66. Thespectral kinetic scan of the 1:21 and 1:31 substrates was found to bewithin background noise for the particular equipment employed.

Now turning to FIG. 4, there is shown a cross-sectional drawing of apresently preferred opaque solid, carbon particles, dispersed within thematrix of a polyamide phase inversion microporous membrane support.Surface (A) illustrates the normal surface of a nylon phase inversionmembrane support, while surface (B) illustrates a cross-sectional viewof such membrane support showing embedded carbon particles within thesolid nylon matrix.

EXAMPLE 1

A coating of fluorescent dye (fluorescein which is a yellow fluorone dye(hydroxylated xanthene)) was air dried on two nylon-66 plates, onefilled with carbon black, the other having no carbon black. The coatedplates were scanned on a luminescence spectrometer.

These spectra indicated that the nylon-66 plate which did not containcarbon black emitted a broader band shape, suggesting that fluoresceinemitted within the same region as the nylon-66. The carbon black fillednylon-66 plate, however, indicated that the energy was lower andslightly shifted to lower wavelengths (probably due to the coatingmatting the reflective energy of the plate surface).

A diluted sample of fluorescein in water was also pre-scanned on theluminescence spectrometer. The fluorescein spectrum indicated that theemitted energy was located near the wavelength found in the spectrum ofthe carbon black filled nylon-66 coated fluorescein plate.

In toto, such findings show that carbon black significantly quenches thenylon-66 background emission of energy, and fluorescence.

EXAMPLE 2

A dope formulation comprising about sixteen percent (16%) by weightNylon-66 (Monsanto® Vydyne™ 66Z), about seventy-seven percent (77%) byweight formic acid, and about seven percent (7%) by weight methanol, wasproduced using the methods disclosed in U.S. Pat. Nos. 3,876,738 and4,645,602, the disclosure of each is herein incorporated in theirentirety by reference. This is the standard formulation and method usedto produce the (white) control membrane.

To produce the carbon black-containing membranes of the present example,the method is similar, but altered by adding the carbon black prior tothe addition of Nylon to the solvent. Specific final compositions forthe dopes produced in this example, expressed in % by weight for eachcomponent are shown in Table 1A.

Briefly, the altered method consisted of the following steps: liquidcomponents formic acid and methanol were combined and allowed to reactcompletely in a closed mixing container. After combining the formic acidand methanol, carbon black was added to the mixture prior to addition ofthe Nylon-66 at a weight ratio as shown in Table 1B. This wasaccomplished by opening the closed container and adding the requiredamount of carbon black directly to the liquid dope solvent mixture.Then, Nylon-66 was added to the mixture and the resulting compositionwas rolled in a jar mill to a maximum temperature of about thirty-fourdegrees Celsius (34° C.) in a constant temperature bath using a TechneC-85D constant temperature water recirculator, until all nylon wasdissolved. The jar was removed from the jar mill. A cap with a sealingarrangement for a propeller shaft was fabricated to minimize volatilelosses, and fitted on the jar. The dope was then mixed with a one andone quarter inch three bladed marine propeller attached to a T-line®Model # 134-1 laboratory mixer in the same vessel, in an attempt tothoroughly disperse the carbon particles. This second mixing stepcontinued for about 1 hour at about 450 RPM.

A small portion (approximately 20 ml) of the dope was subsequently castand quenched in a laboratory apparatus to simulate the casting processdescribed in U.S. Pat. No. 3,876,738, to produce a single layer,non-reinforced microporous nylon membrane approximately 5 mils inthickness while wet. The membrane was subsequently washed in deionizedwater, folded over onto itself (to form a structure of approximately 10mils wet) and dried under conditions of restraint to prevent shrinkagein either the machine direction (x-direction) or cross direction(y-direction). The membrane was found to be strong enough physically towithstand further processing (rinsing, drying, handling, etc), much thesame as membrane without carbon added. When the membrane was rubbedvigorously, or when an adhesive tape was applied and removed, no carbonwas displaced except that which was trapped in nylon pieces that werephysically damaged and removed. Substantially all carbon remainedintimately bound to the nylon matrix.

A small sample of dried, double-layer, non-reinforced nylon membranehaving a combined thickness of about eight (8) mils after shrinkage(z-direction, after the collapsing wet pore structure was complete) wasobtained on which a number of physical measurements were made, asfollows:

An initial bubble point (“IBP”) and foam-all-over-point (“FAOP”) wasmeasured, as described in U.S. Pat. No. 4,645,602, using deionized wateras a wet fluid. Mean flow pore (“MFP”) tests were undertaken as in ASTMF316-70 and ANSI/ASTM F316-70. Water flow rate measurements of thenon-reinforced microporous nylon membrane were performed as described inU.S. Pat. No. 4,473,475. Dry membrane thickness was measured with a 12inch diameter platen dial thickness indicator gauge (accuracy ±0.05 mils(=0.00005 inches)). Fluorescence of the membrane was measured on aPerkin-Elmer LS50B Luminance Spectrophotometer with excitation/emissionset at 290/320 nm respectively (excitation/emission slits both set at2.5 nm). The L (lightness) value was determined using a Macbeth Coloreye3100 colorimeter. The L-value is part of the CIE L*a*b* standard forcolorimetric analysis, one hundred (100) being pure white, and zero (0)being total black. The L-value provides a useful measurement of shadesof gray.

MFP, IBP, FAOP and flow were seen to change with the addition of carbonto the formulation in a manner not directly correlatable with theincrease in carbon concentration. It is believed that a directcorrelation was not seen due to differences in heat build-up duringmixing of the dope. It is known that the structure of the dope can bechanged by temperature increases above the original formulationtemperature (See, U.S. Pat. No. 6,056,529, issued May 2, 2000, thedisclosure of which is hereby incorporated by reference). Fluorescenceintensity, on the other hand, was correlatable to the concentration ofcarbon particles in the substrate. A 1:52 carbon:nylon mix substrate wasfound to exhibit approximately 82.84% less fluorescence than a standardwhite nylon membrane. A 1:15 carbon:nylon mix substrate was found toexhibit approximately 93.13% less fluorescence than a standard whitenylon membrane. TABLE 1A COLOR FORMIC METHANOL NYLON CARBON Carbon:NylonWhite (0:100) % by weight 76.94% 7.08% 15.98% 0.00% Gray (1:52) % byweight 76.90% 6.87% 15.92% 0.31% Black (1:15) % by weight 76.35% 6.80%15.79% 1.06%

TABLE 1B COLOR THICK- WHITE- FLUORES- (Carbon: MFP IBP FAOP NESS FLOWNESS ENCE Nylon) (micron) (psig) (psig) (mils) (ml/min) (L-value)(intensity) White 0.434 43.5 48.0 7.7 49.1 98.15 0.75 (0:100) Gray 0.49035.0 42.0 8.0 82.0 59.11 0.13 (1:52) Black 0.334 48.0 55.0 9.3 35.535.94 0.05 (1:15)

A two by four centimeter piece was subsequently removed from each of thewhite, black and gray membranes above. The tip of a nine-inch Pasteurpipette was heated and stretched to reduce the diameter of the tip. Thiswas then used to spot the membranes with a solution containing 0.1 gramsof 1% by weight aqueous mixture of fluorescent microspheres (fluoresceinlabeled uniform microspheres obtained from Bangs Laboratories, Inc.,catalog # FC03F) with an approximately three-micron diameter in tenmilliliters of distilled water. Under observation, the spots were about250 microns in diameter. The samples with the fluorescent microsphereswere then brought into a darkroom, and illuminated with a hand heldultraviolet light source. With regard to background intensity, the whitemembrane itself glowed brightly, whereas the gray membrane was muchmuted and just slightly visible, and the black membrane disappearedentirely. In each case, the spots placed upon the top of the membranesglowed very well.

EXAMPLE 3

Bacteriophage lambda DNA was digested with Hind III and resolved througha 1.0% agarose gel for 2 hr in Tris-acetate EDTA buffer (40 mMTris-acetate, 1 mM EDTA). Twenty (20) ng, 4 ng, and 0.8 ng samples ofthe digested lambda DNA were run in three separate lanes (demarcated atFIG. 3 as “1, 2, 3”). Three lane “sets” were then transferred toindividual nylon membranes containing different amounts of carbon black(demarcated at FIG. 3 as A-H).

The gels were processed in the following way prior to transfer to thenylon membranes. The gels were soaked with agitation in 500 mL of 0.13 MHCl for 10 minutes. Gels were then transferred into 500 mL 0.5 MNaOH/1.5 M NaCl for 30 minutes. The gels were then neutralized with 0.5M Tris-HCl/1.5 M NaCl for 30 minutes. The gels were then inverted ontosheets of Whatman® 3MM® grade chromatography paper wicks that hang intoa chamber containing 800 mL of standard 10×SSC. Appropriate nylonmembranes were placed on the gel and a stack of 3MM® paper and papertowels were placed on top. A weight, ˜500 g, was placed on top of thestack to facilitate transfer. The transfer was allowed to proceedovernight.

The next day membranes were heated at 80° C. for 60 minutes to dry themand “fix” the nucleic acid to the nylon membrane. Blots werepre-hybridized in 5×SSC with Denhardt's solution at the appropriatetemperature for a minimum of 30 minutes.

Lambda digested with Hind III was radioactively labeled following theprocedure outlined in the MegaPrime Kit from Amersham Pharmacia Biotech.The heat-denatured probe was then added to the blots in 5×SSC/Denhardt'ssolution and hybridizations were carried out overnight at theappropriate temperature.

The following morning the blots were washed in solutions of decreasingionic strength and increasing temperature (stringency washes) to removenon-specific signal. The blots were wrapped in plastic film and exposedto X-ray film in exposure cassettes containing intensifying screens.

FIG. 3 illustrates the results of such experiment with respect to anylon membrane having a carbon black:nylon ratio of (A) 1:10(weight:weight); (B) 1:7.5 (weight:weight); (C) 0:100 (weight:weight)(i.e., control), (E) 1:7.5 (weight:weight) (i.e., repeat of (B)); (F)1:5 (weight:weight), and (G) 0:100 (weight:weight) (i.e., controlrepeat). Also illustrated are the results seen on a commerciallyavailable product, (D) and (H), Amersham® Hybond N+®.

It can be seen that the carbon membrane (tested at 3 differenthigh-level carbon black loadings) successfully binds nucleic acid whencompared to: a standard, unreinforced membrane, (C) and (G), composed ofthe same “casting dope” without added carbon black and to a commerciallyavailable nylon membrane, Amersham Inc., Hybond N+®, (D) and (H), widelyutilized in Southern Blot applications.

From this data, it is also clear that the bound nucleic acid isavailable for hybridization with the radiolabelled probe; thus the addedcarbon black did not affect the accessibility of the DNA forhybridization techniques, nor the efficiency of binding of DNA to Nylon.

EXAMPLE 4

A sample of white membrane (carbon:nylon ratio=0:100), and a sample ofcarbon black impregnated membrane (carbon:nylon ratio=1:10), wereprepared by the method described in Example 2 (the only difference beingthe ratio of carbon black to nylon used in the formulation). Thesesamples were then submitted for cross sectional Scanning ElectronPhotomicrographs.

The particular grade of carbon black which was used in this, and allother examples set forth above, was Manufacturer Degussa-Huls, gradePrintex U Channel black (control number 990225), CAS # 1333-86-4.Channel black typically has a nominal particle size from about 120 toabout 200 angstroms before congealing. Congealed particles can besubstantially larger. Actual particle size has not been measured. It ispreferable to transfer and mix the carbon black in a manner thatminimizes clumping, which will affect the uniformity of dispersionwithin the nylon. For this reason, the second, more vigorous, mixingstep has been employed using a low shear mixing head, such as describedin Example 2. Such mixing equipment is readily available in the market;for example, Lightning® mixers, or other propeller or turbine basedstirring mixers. Such equipment and techniques for mixing can be readilyadapted for these purposes by one skilled in the art.

The white membrane (carbon:nylon ratio=0:100), and carbon blackimpregnated membrane (carbon:nylon ratio=1:10) samples were filled witha neutral media, frozen, then cut using a microtome device in crosssection, which opened up the internal structure of the nylon matrix.After thawing, the neutral media was removed and samples were preparedfor SEM by normal technique. FIG. 5 a shows the white membrane,containing no carbon black (magnification=10,000×). FIG. 5 b shows theinternal structure of the nylon, wherein the impregnated carbon blackparticles are readily visible as inclusions within the nylon matrix(magnification=10,000×). These SEM photos demonstrate the impregnationand substantially wholly incorporated carbon particles in the polyaminesupport, as illustrated in FIG. 4.

In the representative embodiment shown in FIG. 5 b, substantially all ofthe carbon black particles are embedded within the nylon matrix, visibleonly through the area, which has been sliced. There are few if anyvisible particles at or near the (natural, uncut) internal pore surfacesthat can be viewed in the FIG. 5 b SEM photo. Such distribution suggeststhat the internal pore surfaces of the nylon are not chemically affectedby the presence of the carbon black particles.

EXAMPLE 5

A dope formulation comprising about sixteen percent (16%) by weightNylon-66 (Monsanto® Vydyne™ 66Z), about seventy-six percent (76%) byweight formic acid, and about eight percent (8%) by weight methanol, wasproduced using the methods disclosed in U.S. Pat. Nos. 3,876,738 and4,645,602, the disclosure of each is herein incorporated by reference.This is the standard formulation and method used to produce the (white)control membrane.

To produce the carbon black-containing membranes of the present example,the method is similar, but altered by adding the carbon black prior tothe addition of Nylon. Specific final compositions for the dopesproduced in this example, expressed in % by weight of each component areshown in Table 2A.

Briefly, the altered method consisted of the following steps: liquidcomponents formic acid and methanol were combined in a Silverson® Model# L4SRT \SU (Sealed Unit) mixer using a one liter stainless steel vesselat about 1000 rpm for 15 minutes and allowed to react in the closedcontainer. After mixing the formic acid and methanol, carbon was addedto the solution prior to addition of the Nylon-66 at a weight ratio asshown in Table 2A. This was accomplished by opening the closed containerand adding the required amount of carbon black directly to theformic/methanol solution. The carbon was then dispersed with theSilverson® for about 10 minutes at about 5000 rpm. This was done tocompletely disperse the carbon black and facilitate its uniformdistribution throughout the mixture. Then, Nylon-66 was added to themixture and the resulting composition was mixed with a three inchdiameter three-blade propeller mounted on a T-line® Model # 134-1laboratory mixer in the same vessel. A cap with a sealing arrangementfor the propeller shaft was fabricated to minimize volatile losses. Thevessel and mixer arrangement was then placed over a constant temperaturebath such that the majority of the vessel was submerged in the bath,with bath temperature controlled at 34° C. The prop mixer speed was setinitially at about 350 rpm for one-half hour; it was then slowed toabout 70 rpm for another 3 hours to completely dissolve the nylon intosolution. After the three hour mixing, the speed of the mixer was againincreased to about 350 rpm for about one-half hour to completelyhomogenize the dope.

A small portion (approximately 20 ml) of the dope was subsequently castand quenched in a laboratory apparatus to simulate the casting processdescribed in U.S. Pat. No. 3,876,738, to produce a single layer,non-reinforced microporous nylon membrane approximately 5 mils inthickness while wet. The membrane was subsequently washed in deionizedwater, folded over onto itself (to form a structure of approximately 10mils wet) and dried under conditions of restraint to prevent shrinkagein either the machine direction (x-direction) or cross direction(y-direction). The membrane was found to be strong enough physically towithstand further processing (rinsing, drying, handling, etc), much thesame as membrane without carbon added. When the membrane was rubbedvigorously, or when an adhesive tape was applied and removed, no carbonwas displaced except that which was trapped in nylon pieces that werephysically damaged and removed. Substantially all carbon remainedintimately bound to the nylon matrix. By visual (macro) inspection forcolor uniformity across the sheet, and microscopic inspection forevidence of clumping, it appeared that the carbon dispersion of thepresent example was more uniform than in Example 2. This is presentlybelieved to be a result of the high-shear dispersion mixing of carbonparticles in the solvent prior to the addition of the Nylon.

A small sample of dried, double-layer, non-reinforced nylon membranehaving a combined thickness of about eight (8) mils after shrinkage(z-direction shrinkage, after the wet pore structure collapse upondrying was complete) was obtained on which a number of physicalmeasurements were made, as follows:

An initial bubble point (“IBP”) and foam-all-over-point (“FAOP”) weremeasured, as described in U.S. Pat. No. 4,645,602, using deionized wateras a wet fluid. Dry double layer membrane thickness was measured with a½ inch diameter platen dial thickness indicator gauge (accuracy ±0.05mils=0.00005 inches). The L (lightness) value was determined using aMacbeth Coloreye 3100 calorimeter. The L-value is part of the CIE L*a*b*measurement for colorimetric analysis, one hundred (100) being purewhite, and zero (0) being total black. The L-value provides a usefulmeasurement of shades of gray. TABLE 2A (Formulations) COLORCarbon:Nylon FORMIC METHANOL NYLON CARBON White (0:100) 76.14% 7.95%15.91% 0.00% % by weight Gray (1:83) 75.87% 8.09% 15.85% 0.19% % byweight Black (1:15) 75.13% 8.18% 15.69% 1.00% % by weight

TABLE 2B Thickness double Carbon/Nylon IBP FAOP layer Whiteness Content(psig) (psig) (mils) L value White  0:100 67 77 8 98 Gray 1:83 70 80 863 Black 1:15 61 65 9 39

The descriptive use of the color “Gray” in Tables 2A and 2B is only tosuggest that the specific color developed in this example appears to thehuman eye to be gray in comparison to white and black. Clearly, the useof the terms “white” and “black” are similarly flawed. A more precisemeasurement is the “L” value, which has been previously described, andis a value on a continuous, unitless comparison scale.

It is expected that, with routine experimentation, the full range ofwhite to gray to black colors at a full range of useful pore sizes,thickness, etc., may be realized by manipulation of formulae, mixingconditions, and casting conditions familiar to those skilled in the art.Such formulae and mixing conditions will need to be optimized to ensurerobustness, uniformity, and reproducibility of the product. We havedemonstrated here that with simple manipulations of formulae and the useof proper dispersion mixing that it is possible to produce andcharacterize such products.

It is expected that, with routine experimentation, optimal results forhigh sensitivity and low background fluorescent noise can be obtained bysimple manipulation of the variables already disclosed, e.g., pore size,total opaque solids, thickness, etc., and/or other variables in membraneproduction which are familiar to those skilled in the art of membranescience.

While the disclosure has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the disclosurewithout departing from the spirit or scope of the disclosure as definedby the appended claims. All references cited in this specification areherein incorporated by reference to the same extent as if eachindividual reference was specifically and individually indicated to beincorporated by reference.

1-25. (canceled)
 26. An assay in which the presence or quantity of ananalyte is being detected by fluorescence at an emission waveband oflight that results from the excitation of a fluorescent signal label ona polyamide support by a excitation waveband of light, the assaycomprising: a polyamide support; and a plurality of opaque solidsincorporated into the polyamide support, said opaque solids beingsubstantially chemically non-reactive with the polyamide support and asize sufficient to be partially or completely within, or irreversiblybound, to the polyamide support, and quenching fluorescence due to thepolyamide substrate at the excitation waveband or emission waveband, orboth.
 27. The assay as recited in claim 26 wherein the polyamide supportis in the form of a membrane.
 28. The assay as recited in claim 26wherein the opaque solids are carbon particles.
 29. The assay as recitedin claim 28 wherein the carbon particles are less than 5 microns insize.
 30. The assay as recited in claim 29 wherein the carbon particlesare substantially uniformly distributed throughout the polyamidesupport.
 31. The assay as recited in claim 28 wherein the carbonparticles are partially incorporated into the polyamide support.
 32. Theassay as recited in claim 28 wherein the carbon particles aresubstantially wholly incorporated into the polyamide support.
 33. Theassay as recited in claim 28 wherein the substrate is charge-modified.34. A method for preparing a substrate comprising a polyamide supportirreversibly associated with opaque solids, such that substantially allof the polyamide surfaces are chemically and functionally available forbinding of analyte; said method comprising the steps of: formulating acasting dope comprising a solvent, one or more non-solvents, the opaquesolids, and polyamide(s); mixing and blending the casting dope to causedissolution of the polyamide and opaque solids; producing an opaquesolids-filled phase inversion of casting dope; casting a thin portion ofthe opaque solids-filled phase inversion casting dope; quenching thecast portion of the opaque solids-filled phase inversion casting dope toform a substrate; and drying the substrate.
 35. The method of claim 34wherein the opaque solids are carbon particles.
 36. The method of claim35 wherein the carbon particles are less than 5 microns in size.
 37. Themethod of claim 35 wherein the carbon particles are substantiallyuniformly distributed throughout the polyamide support.
 38. The methodof claim 35 wherein the carbon particles are partially incorporated intothe polyamide support.
 39. The method of claim 35 wherein the carbonparticles are substantially wholly incorporated into the polyamidesupport.
 40. The method of claim 34 wherein the polyamide support ischarge-modified. 41-57. (canceled)
 58. A method for preparing asubstrate comprising the acts of: formulating a dope comprising asolvent, at least one non-solvent, opaque solids and at least onephase-inversion polyamide; mixing and blending the dope to causedissolution of the polyamide and opaque solids in the dope; andproducing an opaque solids-filled phase inversion membrane from thedope.
 59. The method of claim 58 wherein the membrane producing actfurther comprises the acts of: casting the dope; quenching the opaquesolids-filled phase inversion dope to form a substrate; and drying thesubstrate.
 60. The method of claim 59 wherein the membrane producing actfurther comprises the acts of: irreversibly associating the membranewith the opaque solids such that substantially all of the membranesurfaces are chemically and functionally available for binding ofanalyte. 61-62. (canceled)